Although significant advances have been made in cancer treatment, the prognosis for oral cancer remains poor in comparison to other cancer types, including breast, skin, and prostate. Therefore, new therapeutic approaches are necessary to improve the outcome of this disease. The discovery that the introduction of chemically synthesized small interfering RNAs (siRNAs) into mammalian cells could efficiently induce sequence-specific inhibition of gene expression, made evident the therapeutic potential of harnessing RNA interference (RNAi) as a means to specifically target and silence disease-causing genes. Although the design of therapeutic-grade siRNAs has improved, delivery still remains the single greatest obstacle towards the pervasive use of siRNAs for therapeutic applications. Thus, to enhance the intracellular bioavailability of siRNAs in diseased tissues, effective new strategies for delivery are needed. Recently, we demonstrated that a novel peptide carrier we designed, termed 599, that comprised cell-penetrating and endosome-disruptive properties, could enhance the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncogene (siCIP2A) into oral cancer cells in vitro, with intratumoral administration of the 599 peptide-siCIP2A complex inducing CIP2A silencing and consequently tumor growth inhibition in vivo. Despite these findings, further studies are still needed to better understand the importance in the chemical design of the 599 peptide and in determining the therapeutic effectiveness of administering the 599 peptide intravenously, since systemic delivery remains the standard method of administering drugs for the treatment of solid tumors. Consequently, because the 599 peptide was not cell/tissue-specific, we recently developed a dual peptide-mediated technology, where we found in our preliminary studies that combining a cancer cell-targeting peptide with the 599 peptide into a dual peptide-siCIP2A complex could mediate increased targeted delivery of siCIP2As into tumor tissues and significantly enhance CIP2A silencing upon systemic delivery. Therefore, based on the above findings, the goal of the current proposal is to further develop and improve upon the design of this 599 peptide-based siRNA delivery strategy for the treatment of oral cancer by determining the importance of amino acid chirality in 599 peptide design/function and whether systemic delivery of siCIP2As using the dual peptide- mediated technology can reduce tumor burden. To accomplish this goal, we will use biochemical and molecular biology approaches to: (1) determine the functional importance of stereochemistry in terms of 599 peptide-mediated intracellular delivery of bioactive siRNAs; and (2) determine the efficacy of the dual peptide- siCIP2A complex in mediating tumor growth inhibition in treated animals. The outcomes of the proposed research are expected to lead to improved chemical designs of the 599 peptide in mediating the delivery of bioactive siRNAs into cancer cells/tissues and in helping establish the therapeutic potential of the dual peptide- mediated technology for RNAi-based therapy in human oral cancer.